Project Summary Cryptococcus neoformans is an encapsulated budding yeast that causes a life-threatening illness in immunocompromised individuals, especially in AIDS patients. Although the infection starts in the lung, cryptococcosis commonly presents as meningoencephalitis, which is one of the most common infections of the central nervous system and a leading cause of death in HIV-infected individuals. Transmigration of C. neoformans across the blood-brain barrier (BBB) is believed to be one of the most critical steps in the development of cryptococcal meningoencephalitis. In vitro studies have shown that C. neoformans can transmigrate across a monolayer of brain microvascular endothelial cells (BMECs) through transcytosis and ?Trojan horse? pathways. However, in vivo the BBB is a complex tissue that consists of BMECs, pericytes, astrocyte end feet, and a basement membrane in a precise organization, which cannot at present be recreated in vitro. Questions still remain as to how C. neoformans migrates to the brain across the BBB in vivo and what is the underlying mechanism(s). Answering these questions is fundamental for understanding cryptococcal pathogenesis, because brain invasion is the hallmark feature of this disease and meningoencephalitis is the major and most lethal complication of cryptococcosis. In contrast to previous in vitro studies that did not account for vascular haemodynamics, we have developed a novel in vivo model system based on multiple novel approaches to directly investigate the brain invasion by C. neoformans in vivo. In this project, we will use this in vivo system to study traversal of the BBB by C. neoformans by addressing the following aims: 1) To characterize the transcytosis of C. neoformans and the mechanism underlying this pathway in vivo. 2) To investigate the ?Trojan horse? pathway and the underlying mechanism during brain infection in vivo. 3) To characterize the mechanism(s) whereby C. neoformans damages the BBB in vivo. 4) To determine the relative contribution of each mechanism to brain invasion by C. neoformans. At the end of the proposed work, we will have identified the in vivo mechanism(s) by which C. neoformans traverses the BBB. The knowledge created in this study will provide new potential therapeutic targets of intervention for this disease.